Nitric oxide and its possible implication in ME/CFS (Part 2 of 2)

Nitric oxide and its possible implication in ME/CFS (Part 2 of 2)Andrew Gladman explores the current and historic hypotheses relating to nitric oxide problems in ME/CFS. This second article in a 2-Part series puts nitric oxide under the microscope and explores what it is, what it does and why it is so frequently discussed in the world of ME/CFS. The focus of this article is to build upon the previous article and explore how the hypotheses surrounding ME/CFS and nitric oxide weigh up against one another.

This is the second part of a previously published article, it is therefore advised to read Part 1, which can be found here, first.

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In the last article we explored what exactly nitric oxide is, how it is produced in the body and the numerous different functions it has over a variety of different bodily systems.The focus of this article is going to be how different hypotheses, specifically a dysfunctional production of nitric oxide, has the potential to explain the primary symptoms of ME/CFS. During this process we will also pick out some of the flaws such a hypothesis could have.How could nitric oxide and autoimmunity explain the symptoms of ME/CFS?

If the hypotheses relating to insufficient NO production in ME/CFS proves to be correct, the symptoms of ME/CFS could be broken down into three distinct categories:

Firstly there is the symptoms that result from the direct dysregulation of the immune system. These include swollen lymph nodes and the sore throat that many patients experience.

Secondarily there are symptoms that result from a lack of NO production within the endothelial tissue. These likely include heat sensitivity and could potentially explain the ease of muscle fatigability.

Finally there are the symptoms that could result from inefficient NO production in the nervous system. These are potentially the most wide-ranging, including autonomic dysfunction and sleep disruption.

The following is a brief overview of several common ME/CFS symptoms and how inhibition of nNOS and eNOS and potential overstimulation of iNOS relate.

Headache/Migraine:Migraine-type headaches are thought to be a comorbid entity and symptom in as many as 80% of ME/CFS sufferers, this is the highest prevalence of all the symptoms in ME/CFS as Professor Baraniuk stressed in his presentation at the IiME conference in 2012.

This comorbidity may therefore be pointing towards a fundamental physiological problem caused by the disease process of ME/CFS, especially in the light of this new hypothesis considering migraines are thought to be a neurovascular condition.

Nitric oxide is nothing new in migraine research and it appears to be the reason why ME/CFS patients appear to get such extreme and common headaches. Not suprising, considering migraines are considered neurovascular in origin, and NO plays a crucial role in both the neurological and vascular systems.

Sleep:Another common symptom ME/CFS patients experience and many suffer with daily is disrupted sleep. The role of nitric oxide in sleep is thought to be through its production by nNOS by neurones within the brain however NO production by iNOS is also thought to potentially play a role in this. NO contained within neurones is thought to be of great importance for initiation and maintenance of sleep, particularly REM sleep.

Nitric oxide acts as the trigger for biogenesis of more mitochondria in cells​

Muscle Function/Post Exercise Malaise:Muscle function and ease of fatigability along with post-exercise malaise is incredibly common in ME patients, and must be present in many diagnostic criteria for a diagnosis of ME/CFS.

The new hypothesis has the potential to explain this symptom. Under normal circumstances, oxygen in the blood diffuses into cells and then into the mitochondria.

It is used during the final phase of aerobic respiration, known as oxidative phosphorylation during which adenosine triphosphate (ATP) is produced. ATP is the molecule known as the universal energy currency in the body.

If eNOS is inhibited, nitric oxide cannot act to dilate the blood vessels in muscle tissue. The lower supply of blood to the peripheral circulation and muscles means that the supply of oxygen and removal of waste materials cannot occur at a sufficient rate in respiring tissues.

This means that when the muscles are used in ME/CFS patients, there is a much lower ability to produce ATP through aerobic respiration. Therefore anaerobic respiration has to 'pick up the slack' to provide the vital ATP required for voluntary muscle contraction.

Anaerobic respiration is much less efficient than aerobic respiration meaning that the lack of energy many patients describe themselves as having could indeed be due to a lowered ability to produce ATP at a normal, sufficient rate.

This anaerobic respiration also produces lactic acid as a byproduct. Given the inability of the blood vessels to dilate as required, lactic acid remains in the tissues and cells for much longer than required, likely causing the muscle fatigue and prolonged adverse effect to exercise, as professor Julia Newton discovered when researching muscle abnormalities in ME/CFS.

Furthermore the dysregulation and lower number of mitochondria due to the absence of NO to trigger mitochondrial biogenesis only further reduces the ability of mitochondria to produce ATP through aerobic respiration.

ATP - the universal energy currency in the cell, most of which is produced within the mitochondria​

Under normal circumstances the nitric oxide-controlled potassium channels on the outer membrane of the mitochondria remain closed. However when a lot of ATP is required (so termed a cellular energy crisis) the mitochondrial function actually begins to decrease due to the decline in difference between the two regions of the mitochodrion containing protons (H+).

To combat this decline in efficiency these potassium channels open, allowing positively charged potassium molecules (ions) to move between these two isolated regions of the mitochondria. This restores the electrochemical gradient and allows further ATP production to continue at an optimum rate.

In the absence of nitric oxide these channels cannot open, and as such ATP production by the mitochondria slows drastically. Without nitric oxide there to open these channels the individual cells. Hence tissues, made up from these cells, are incapable of supplying the ATP required during times of high demand. Interestingly, the further this demand stretches the longer it takes the mitochondria to recover, especially when you consider the slower generation of new mitochondria due to lack of NO.

Could this be the cellular basis for post-exercise malaise?

In ME/CFS, there is an insufficient supply of blood to the muscles, meaning that supply of oxygen and removal of waste products such as lactic acid is not sufficient. The mitochondia are dysfunctional with regard to ATP and unable to cope with exercise induced stress, with further stress increasing the degree of dysfunction and increasing mitochondrial recovery time.

Finally, the biogenesis of new mitochondria cannot occur at a sufficient rate due to the lack of nitric oxide to act as the triggering molecule. When combined, these factors result in the intense exercise intolerance, the increased build-up of lactic acid due to more energy having to be derived through anaerobic respiration, and possibly the post-exercise malaise that ME/CFS patients experience.

For further discussion on mitochondria in ME/CFS looking beyond nitric oxide, be sure to check out the recent article published here at Phoenix Rising.

Sensory-sensitive and tremor sensation:

Motor neurone: potassium channels are located the entire length of the neurone, embedded within the cell membrane, serving to limit the duration of nerve impulses (action potentials)​

As discussed previously, neurones within the central and peripheral nervous systems produce nitric oxide for use as a neurotransmitter and signalling molecule. This is achieved through the action of nNOS.

Further to this action however, nitric oxide activates potassium/ATP channels on the surface of the neurone, allowing for the flow of potassium out of the neurone.

The transmission of nerve impulses is in itself a complex topic, involving many channel proteins and the diffusion of charged sodium and potassium molecules (ions) in and out of neurones.

Potassium acts by exiting the neurone via this channel following an influx of sodium. This reduces the duration of the nervous signal (action potential) hence saving energy and preventing the continual firing of nervous signals.

Nitric oxide is like the key which the doorman at a school uses to open the gate for the children to leave, allowing the bustling crowd of students out at the end of the school day. Without it, the students stay locked in the school, so more and more noise builds. There are other smaller 'gates' for the children to exit through but this takes much longer, so the noise takes longer to cease.

This noise allows for prolonged and potentially damaging nervous impulses, which could explain the sensory sensitivity and phenomena many ME/CFS patients suffer, such as light and sound sensitivity and the sensation of internal tremor following overexertion.

Orthostatic intolerance:There is a huge array of evidence and reports of orthostatic intolerance (OI) within ME/CFS. OI is thought to be directly affected through dysregulation of blood pressure. Therefore the links are clear to draw with the lack of endothelial NO production which this new hypothesis alludes to.

This area has several researchers looking into it, including Professor Julia Newton. Prof. Netwon’s work approaches the abnormalities from the perspective of an autonomic nervous system problem, which could also result from this new hypothesis, given the use and production of NO in the nervous system by nNOS.

For example: “The heart MRI scans showed that a third of people with ME/CFS have impaired bioenergetic function in their hearts which has a knock on effect upon the function of their hearts.” This is clearly relatable to this new hypothesis, wherein the impaired function could come as a direct result of inability to correctly dilate blood vessels.

At this point it is important to remember that the endothelial cells form the innermost layer within all blood vessels and hence is in direct contact with the blood stream. It is therefore the easiest location within the vessel for circulating autoantibodies to target.

Temperature homeostasis:This hypothesis also has obvious further implications on correct internal control of temperature. Given the role of NO in dilating blood vessels, vasoconstriction would likely dominate in peripheral circulation.Perhaps this is why many ME/CFS patients describe their symptoms as a little easier to manage in cooler weather.

In a healthy individual, warmer weather generally stimulates vasodilation and thus increases peripheral blood circulation, allowing heat to be lost by conduction through the skin. If this hypothesis is correct, for ME patients this dilation does not occur therefore in warmer weather ME/CFS patients find it much more difficult to cool down and are also much more likely to overheat.

It is fairly well documented that ME/CFS patients suffer with intolerance to extremes in temperature and an autoimmune hypothesis relating to nitric oxide among other hypotheses has the potential to explain why this is so.

The work of Newton et al. at the Univerity of Dundee, specifically their study from February 2012 outlined their discovery that ME/CFS patients have measurable large and small artery endothelial dysfunction relative to age and gender matched healthy controls. This is a very important finding if the data proves repeatable and especially interesting given the important function nitric oxide plays in the cardiovascular system.

Immune Dysfunction:One further sign and symptom I am going to explore is the dysregulation of the immune system that has been reported by many patients and researchers, specifically the action of natural killer cells. Nitric oxide exhibits the ability to regulate the action and activation of NK cells. Given the plethora of evidence implicating NK cell dysfunction in ME/CFS through studies such as those at Griffith University, this raises a potential mechanism through which this may occur.

Researchers have discovered that the NOS expressed by NK cells is in fact eNOS. Given that NO appears to activate NK cells, it is therefore clear that any autoimmune mechanism blocking the production of NO by eNOS and nNOS would therefore retard NK cell activity. This has has been well documented in ME/CFS, in spite of the seemingly up-regulated action of iNOS in phagocytes.

Somewhat recently during the CDC conference call in 2013, Professor Ian Lipkin revealed some preliminary data from his upcoming publication. A part of this as of yet to be published data outlines the findings of many markers of immune dysregulation and activation. Included are many increases and decreases relating to a number of cytokines including Interlukin 17, 2 and 8 and an interesting decrease in TNF.

This further implies that inflammation appears not be a mechanism present within the pathology of ME/CFS, while the other markers of immune dysregulation could lend further credence to the autoimmune hypothesis. It is important, though, to note that Prof. Lipkin himself still believes the pathology to be of an infectious nature.

ConclusionThere are a few problems with these hypotheses, however. This is not the first time that the endothelial dysfunction and nitric oxide production have been implicated within autoimmune diseases. There is evidence of endothelial dysfunction being present in both lupus erythematosus, assorted rheumatic autoimmune diseases, and damage as a consequence of diabetes mellitus. This presents as an increased risk of atherosclerosis and slower wound healing, presumably through the loss of peripheral circulation.

As previously discussed, this increased risk does not at the present time appear to exist within ME/CFS. This prevalence in other diseases does, however, raise the question of whether an immune response targeted towards nitric oxide production could be a secondary mechanism within ME/CFS as seen in these other autoimmune processes.

Over the years, ME/CFS has been the subject of many a debate and has had hundreds of different researchers speculating upon hundreds of different disease causes and mechanisms.

Only recently has the idea of ME/CFS as an autoimmune entity gained any significant and reliable data to support it, yet it appears to represent one of the best research directions for years if not decades. This new focus upon autoimmunity appears to have reignited an ailing research area in nitric oxide. Only time will tell if this particular line of thought leads to anything greater, but the push of research is certainly an inspiring sight to see in such a neglected disease as ME/CFS.

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Currently the prevailing view within ME/CFS research is that there is upregulation of NO production within the immune system, as initially proposed by Pall. This can now be evidenced by upregulation of the NF-KB/iNOS pathway and increased levels of NO-conjugated molecules (Maes et al).

I'm not really aware of there being a hypothesis relating to an autoimmune response against NOS? Although there are several suggestions of low NO levels. This could relate to things like disturbed redox, autonomic dysfunction or autoimmunity at various levels.

Also whilst NO attracts all the attention it is just one of the gaseous mediators, the others being H2S and CO. Hydrogen sulfide (H2S) biology in particular is analogous to NO: H2S is released from both constitutive and inducible enzymes; H2S works with NO to induce vasodilation; immune cell activation requires H2S; and H2S is readily oxidised to sulfite.

Whats interersting is that dr jay goldstein used meds like glycerol trinitrate and viagra, both which can increase NO, im unsure on the theory of why it helped, possibly increasing cerebral perfusion??

Its my understanding that its peroxynitrate that comes from NO thats the bad boy, not NO directly??

Whats interersting is that dr jay goldstein used meds like glycerol trinitrate and viagra, both which can increase NO, im unsure on the theory of why it helped, possibly increasing cerebral perfusion??

Its my understanding that its peroxynitrate that comes from NO thats the bad boy, not NO directly??

Click to expand...

NO is a free radical, but needed in small amounts. ONOO however is hair bleach and just plain nasty. My understanding of Pall's stuff was that he theorised to ,much ONOO, not enough NO.

At last a possible explanation for the heat sensitivity! Seems very plausible. Ditto the effects on oxygen delivery and lactate removal, but those wouldn't have affected Julia Newton's in vitro findings; however, the potassium channel problem and low mito numbers could have.

I seem to be in a minority again re an ME symptom - no migraines and only very mild headaches which are by no means disabling apart from times of worsening and other occasional episodes.

Seems odd that most pwME seem to have hypotension despite apparently-excessive vasoconstriction. I have hypertension but seem to share the common low-blood-volume problem - or do I? (Never had it tested - my doctors seem to prefer presumption and guesswork.)

Another anomaly is that a team including KDM found higher levels of NO in CFS:

I still don't understand if NO is supposedly high or low in ME/CFS...or if it can be accurately measured by those test strips that are sold.

If we assume similarities to MS (which is a leap), it seems that in the presence of low or normal (i.e. non pregnancy) levels of estriol, nitric oxide synthetase is not inhibited which may lead to higher than normal levels of NO due to the stimulation by the inflammatory cytokines.

Have I got that right?

Another possible protective mechanism in this M.S. story may be the inhibition of an enzyme nitric oxide synthetase by estriol which is produced by microglial cells activated by these inflammatory cytokines. See the abstract by Drew and Chavis from University of Arkansas Medical School in Journal of Neuroimmunology 2000.

Abstract
Multiple sclerosis occurs more commonly in females than males. However, the mechanisms resulting in gender differences in multiple sclerosis are unknown.

Activated microglia are believed to contribute to multiple sclerosis pathology, perhaps in part due to production of nitric oxide (NO) and TNF-alpha, molecules which can be toxic to cells including oligodendrocytes.

The current study demonstrates that the female sex steroids estriol, beta-estradiol and progesterone inhibit lipopolysaccharide (LPS) induction of nitric oxide (NO) production by primary rat microglia and by the mouse N9 microglial cell line. These hormones act by inhibiting the production of inducible nitric oxide synthase (iNOS) which catalyses the synthesis of NO.Estriol likely inhibits iNOS gene expression since the hormone blocks LPS induction of iNOS RNA levels.

The pro-inflammatory cytokines IFN-gamma and TNF-alpha are believed to be important modulators of multiple sclerosis. Here, we demonstrate that estrogens and progesterone also inhibit NO production by microglial cells activated in response to these cytokines. Activated microglia elicit TNF-alpha in addition to NO and we further demonstrate that estrogens and progesterone repress TNF-alpha production by these cells.

Finally, estriol and progesterone, at concentrations consistent with late pregnancy, inhibit NO and TNF-alpha production by activated microglia, suggesting that hormone inhibition of microglial cell activation may contribute to the decreased severity of multiple sclerosis symptoms commonly associated with pregnancy.

There is another overlooked element to this hypothesis that should be given some consideration.

If the blood cells are "floppy" due to impaired filling, would the same then also apply to the adhesion molecules complex that forms as a part of the inflammation process. For instance, could dysfunction of P-selectin in particular, which is induced to translocate to the endothelial cell surface with activation of endothelial cells in the presence of oxygen-derived free radicles or ischemia/reperfusion, among other causes, lead to blood staying "sticky" and the issue then becomes one of blood viscosity, rather a volume problem as is often cited?

There is another overlooked element to this hypothesis that should be given some consideration.

If the blood cells are "floppy" due to impaired filling, would the same then also apply to the adhesion molecules complex that forms as a part of the inflammation process. For instance, could dysfunction of P-selectin in particular, which is induced to translocate to the endothelial cell surface with activation of endothelial cells in the presence of oxygen-derived free radicles or ischemia/reperfusion, among other causes, lead to blood staying "sticky" and the issue then becomes one of blood viscosity, rather a volume problem as is often cited?

Interesting idea - I'm not that au fait with the mechanisms referred to, but my blood viscosity has been normal when tested, and it seems to flow normally - health professionals don't usually have much trouble taking blood samples from me.

I still don't understand if NO is supposedly high or low in ME/CFS...or if it can be accurately measured by those test strips that are sold.

If we assume similarities to MS (which is a leap), it seems that in the presence of low or normal (i.e. non pregnancy) levels of estriol, nitric oxide synthetase is not inhibited which may lead to higher than normal levels of NO due to the stimulation by the inflammatory cytokines.

There have been both claims that NO is high, and claims that ONOO is high & NO low. I have still not gotten my head around the techniques for testing blood for NO & ONOO well enough to form a view of the science.

I am still working through this stuff but my understanding is that in general yes Cytokines to stimulate NO production within infected cells damaging both the parasite and the infected cell.

Whats interersting is that dr jay goldstein used meds like glycerol trinitrate and viagra, both which can increase NO, im unsure on the theory of why it helped, possibly increasing cerebral perfusion??

Its my understanding that its peroxynitrate that comes from NO thats the bad boy, not NO directly??

Click to expand...

Glycerol trinitrate, the related isosorbide and Viagra (a phosphodiesterse inhibitor, PDE5) are all NO donors that work through stimulation of cyclic guanosine monophosphate (cGMP) pathway which inhibits calcium influx and also a direct activation of potassium channels leading to hyperpolarization and relaxation of smooth muscle. http://cvpharmacology.com/vasodilator/nitro.htm

Breaking the calcium influx cycle through opening of potassium channels is also what happens with Nicorandil which I posted about back in May. See "Nicorandil.....A role in treating CFS?". I use Nicorandil (nicotinamide nitrate) for pain control in bowel ischemia but also found that the induced vasodilation also improved my symptoms of Gastroparesis, another issue found in a subset of those with ME/CFS.

I think the confusion over NO is related in part to the situation that can arise when too much iNOS is being generated as this can then uncouple eNOS, so too much of one, not enough of the other.

Whats interersting is that dr jay goldstein used meds like glycerol trinitrate and viagra, both which can increase NO, im unsure on the theory of why it helped, possibly increasing cerebral perfusion??

Its my understanding that its peroxynitrate that comes from NO thats the bad boy, not NO directly??

Click to expand...

Heaps,

his stuff is really difficult to understand, but he talks about a 'broken cycle' in which oxidised Nitric Oxide is not converted back to its reduced form. It's also possible due to at least two enzymatic dysfunctions to directly produce too much ONOO (peroxynitrate). My guess would be you need to talk to Alex, he talked directly with Pall about this stuff.